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United States Patent |
6,191,254
|
Falla
,   et al.
|
February 20, 2001
|
Antimicrobial cationic peptides
Abstract
A novel class of cationic peptides having antimicrobial activity is
disclosed. These peptides can be encompassed by the formulas:
X.sub.1 X.sub.1 PX.sub.2 X.sub.3 X.sub.2 P(X.sub.2 X.sub.2 P).sub.n
X.sub.2 X.sub.3 (X.sub.5).sub.o ; (SEQ ID NO: 23)
X.sub.1 X.sub.1 PX.sub.2 X.sub.3 X.sub.4 (X.sub.5).sub.r PX.sub.2
X.sub.3 X.sub.3 ; (SEQ ID NO: 24)
X.sub.1 X.sub.1 X.sub.3 (PW).sub.u X.sub.3 X.sub.2 X.sub.5 X.sub.2
X.sub.2 X.sub.5 X.sub.2 (X.sub.5).sub.o ; and (SEQ ID NO: 25)
X.sub.1 X.sub.1 X.sub.3 X.sub.3 X.sub.2 P(X.sub.2 X.sub.2 P).sub.n
X.sub.2 (X.sub.5).sub.m ; (SEQ ID NO: 26)
wherein:
m is 1 to 5;
n is 1 or 2;
o is 2 to 5;
r is 0 to 8;
u is 0 or 1;
X.sub.1 is Isoleucine, Leucine, Valine, Phenylalanine, Tyrosine, Tryptophan
or Methionine;
X.sub.2 represents Tryptophan or Phenylalanine
X.sub.3 represents Arginine or Lysine;
X.sub.4 represents Tryptophan or Lysine; and
X.sub.5 represents Phenylalanine, Tryptophan, Arginine, Lysine, or Proline.
The invention also provides a method of producing a cationic peptide
variant having antimicrobial activity.
Inventors:
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Falla; Timothy J. (Vancouver, CA);
Hancock; Robert E. W. (Vancouver, CA);
Gough; Monisha (Vancouver, CA)
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Assignee:
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University of British Columbia (Vancouver)
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Appl. No.:
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702054 |
Filed:
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August 23, 1996 |
Current U.S. Class: |
530/300; 530/324; 530/326; 530/327 |
Intern'l Class: |
A61K 038/00; A61K 038/04 |
Field of Search: |
530/300,324,326,327
514/12,13
424/185.1
|
References Cited
U.S. Patent Documents
4810777 | Mar., 1989 | Zasloff.
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4822608 | Apr., 1989 | Benton et al.
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5028530 | Jul., 1991 | Lai et al.
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5073542 | Dec., 1991 | Zasloff.
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5166321 | Nov., 1992 | Lai et al.
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5202420 | Apr., 1993 | Zasloff et al.
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5206156 | Apr., 1993 | Lai et al.
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5208220 | May., 1993 | Berkowitz.
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5217956 | Jun., 1993 | Zasloff et al.
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5235038 | Aug., 1993 | Blondelle et al.
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5254535 | Oct., 1993 | Zasloff et al.
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5324716 | Jun., 1994 | Selsted et al. | 514/14.
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5344765 | Sep., 1994 | Lai et al.
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5357044 | Oct., 1994 | Lai et al.
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5547939 | Aug., 1996 | Selsted | 514/14.
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5635594 | Jun., 1997 | Lehrer et al. | 530/317.
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WO9406688 | Mar., 1994 | WO.
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WO 95/22338 | Aug., 1995 | WO.
| |
Other References
"Biologically Active and Amidated Cecropin Produced in a Baculovirus
Expression System From a Fusion Construct Containing the Antibody-Binding
Part of Protein A" The Biochemical Journal 280, Part 1: 219-224, 1991.
Romeo et al., "Bovine Neutrophil Antibiotic Peptides and Their Precursors:
Structure and Role Innate Immunity," Croatica Chemica ACTA, vol. 68, No.
3, 1995 pp. 607-614.
Van Abel et al., "Synthesis and characterization of indolicidin, a
tryptophan-rich antimicrobial peptide from bovine neutrophils", Int. J.
Peptide Res.; 45, 1995, 401-409.
Uchida et al., "Antibacterial Activity of the Mammalian Host Defense
Peptide, Indolicidin, and Its Fragments," Peptide Chemistry, 1995, N.
Nishi (Ed.) (1996), pp. 229-232.
REW Hancock et al. In: Molecular Biology of Pseudomonads, International
Symposium on Pseudomonads, 5th Meeting, Mol. Biol. Biotechnol., 1995 (Ed)
T. Nakazawa et al. Chapter 38, pp. 441-450, 1996 ASM Press, Washington,
DC.
J. Rudinger. In: Peptide Hormones, (Ed) JA Parsons et al. pp. 1-7,
University Park Press, Baltimore, 1976.
E. Lazar et al. Mol. Cellular Biol. 8(3): 1246-1252, 1988.
WH Burgess et al. J. Cell Biol. 111: 2129-2138, 1990.
TJ Fall et al. J. Biol. Chem. 271(32): 19298-19303, 1996.
C. Subbalakshmi et al. FEBS Lett. 395: 48-52, 1996.
Del Sal et al. Biochem. Biophys. Res. Commun. 187: 467-472, abstract, 1992.
Selsted et al. J. Biol. Chem. 67: 429-495, abstract, 1992.
|
Primary Examiner: Housel; James C.
Assistant Examiner: Devi; S.
Attorney, Agent or Firm: Gray Cary Ware & Freidenrich LLP, Haile; Lisa A.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATION
This Application claims the priority of U.S. Provisional Application Ser.
No. 60/002,687, filed Aug. 23, 1995, incorporated herein by reference.
Claims
What is claimed is:
1. An isolated cationic peptide selected from the group consisting of:
TBL
ILKKWPWWPWRRK (SEQ ID NO: 1);
ILKKWPWWPWRR (SEQ ID NO: 5);
LPWKWPWWPWRKWR (SEQ ID NO: 6);
LPWKWPWWPWRRWR (SEQ ID NO: 7);
LPWKWPWWPWWPWRR (SEQ ID NO: 11);
LPWKWPWWPWWKKPWRR (SEQ ID NO: 12);
LKKWPWWPWKWKK (SEQ ID NO: 18);
LKKFPFFPFRRK (SEQ ID NO: 28); and
FKKFPFFPFRRK (SEQ ID NO: 36).
2. An isolated cationic peptide selected from the group consisting of:
TBL
LKKWPWWPWKRR (SEQ ID NO: 17);
LKKWPWWRWRR (SEQ ID NO: 27);
LKKFPFFPFKKK (SEQ ID NO: 29);
LKKWAWWPWRRK (SEQ ID NO: 30);
LKKWPWWAWRRK (SEQ ID NO: 31);
LKKWPWWPWKKK (SEQ ID NO: 32);
LRRWPWWPWRRR (SEQ ID NO: 33);
[WKKWPWWPWRRK (SEQ ID NO: 34)];
FKKWPWWPWRRK (SEQ ID NO: 35);
FKKFPFFPFKKK (SEQ ID NO: 37);
ILKKWPWWPWWPWRRK (SEQ ID NO: 38);
ILKKWPWWPWRWWRR (SEQ ID NO: 39);
ILKKWPWWPWRRWWK (SEQ ID NO: 40);
ILKKWPWWPWPPRRK (SEQ ID NO: 41); and
ILKKWPWWPWPPFFRRK (SEQ ID NO: 42).
3. An isolated cationic peptide consisting of an amino acid sequence
selected from the group consisting of:
TBL
ILPWICPWRPSKAN (SEQ ID NO: 13);
IVPWKWTLWPWRR (SEQ ID NO: 14); and
TLPCLWPWWPWSI (SEQ ID NO: 15).
4. An isolated cationic peptide having an amino acid sequence selected from
the group consisting of:
TBL
ILKPWKWPWWPWRRKK (SEQ ID NO: 2);
ILKPWKWPWWPWRR (SEQ ID NO: 3);
ILPWKKWPWWRWRR (SEQ ID NO: 4);
ILPWKWPWRR (SEQ ID NO: 10);
ILPWKWPWYVRR (SEQ ID NO: 19);
IKWPWYVWL (SEQ ID NO: 20);
ILPWKWFFPPWPWRR (SEQ ID NO: 21); and
LPWKWPPWPPWPWRR (SEQ ID NO: 22).
5. A peptide of claim 1, 2, 3 or 4, wherein the peptide is amidated.
6. A peptide of claim 1, 2, 3 or 4, wherein the peptide is
carboxymethylated.
Description
FIELD OF THE INVENTION
The invention relates to peptides that have antimicrobial activity.
BACKGROUND OF THE INVENTION
Systemic diseases that are associated with pathogenic microorganisms or
their toxins in the blood (e.g., septicemia) are a leading cause of death
among humans. Gram-negative bacteria are the organisms most commonly
associated with such diseases, and pathogenesis has been related in many
cases to the release of a toxic outer membrane component termed endotoxin.
However, gram-positive bacteria are an increasing cause of fatal
infections. In addition, antibiotic resistance is becoming a major problem
for all classes of antibiotics, and novel antibiotics are urgently needed.
Cationic peptides having antimicrobial activity have been isolated from a
wide variety of organisms. In nature, such peptides provide a defense
mechanism against microorganisms such as bacteria and yeast. Generally,
these cationic peptides are thought to exert their antimicrobial activity
on bacteria by interacting with the cytoplasmic membrane to form channels
or lesions. In gram-negative bacteria, they interact with surface
lipopolysaccharide (LPS) to permeabilize the outer membrane, leading to
self promoted uptake across the outer membrane and access to the
cytoplasmic membrane. Examples of antimicrobial peptides include
indolicidin, defensins, cecropins, and magainins.
SUMMARY OF THE INVENTION
The invention provides a novel class of isolated cationic peptides having
antimicrobial activity. As a group, the peptides of the invention can be
described by the following formulas:
X.sub.1 X.sub.1 PX.sub.2 X.sub.3 X.sub.2 P(X.sub.2 X.sub.2 P).sub.n
X.sub.2 X.sub.3 (X.sub.5).sub.o ; (SEQ ID NO: 23)
X.sub.1 X.sub.1 PX.sub.2 X.sub.3 X.sub.3 X.sub.4 (X.sub.5).sub.r
PX.sub.2 X.sub.3 X.sub.3; (SEQ ID NO: 24)
X.sub.1 X.sub.1 X.sub.3 (PW).sub.u X.sub.3 X.sub.2 X.sub.5 X.sub.2
X.sub.2 X.sub.5 X.sub.2 (X.sub.5).sub.o ; and (SEQ ID NO: 25)
X.sub.1 X.sub.1 X.sub.3 X.sub.3 X.sub.2 P(X.sub.2 X.sub.2 P).sub.n
X.sub.2 (X.sub.5).sub.m ; (SEQ ID NO: 25)
wherein:
m is 1 to 5;
n is 1 or 2;
o is 2 to 5;
r is 0 to 8;
u is 0 or 1;
X.sub.1 is Isoleucine, Leucine, Valine, Phenylalanine, Tyrosine, Tryptophan
or Methionine;
X.sub.2 represents Tryptophan or Phenylalanine;
X.sub.3 represents Arginine or Lysine;
X.sub.4 represents Tryptophan or Lysine; and
X.sub.5 represents Phenylalanine, Tryptophan, Arginine, Lysine, or Proline.
Various derivatives, analogues, conservative variations, and variants of
the peptides described herein are included within the invention.
Preferably, the peptide is amidated or carboxymethylated. Isolated nucleic
acids encoding the peptides of the invention also are included. Examples
of preferred peptides include those having the following amino acid
sequences, as defined using the one-letter amino acid code:
ILKKWPWWPWRRK (SEQ ID NO: 1);
ILKPWKWPWWPWRRKK (SEQ ID NO:2);
ILKPWKWPWWPWRR (SEQ ID NO:3);
ILPWKKWPWWRWRR (SEQ ID NO:4);
ILKKWPWWPWRR (SEQ ID NO:5);
ILPWKWPWWPWRKWR (SEQ ID NO:6);
ILPWKWPWWPWRRWR (SEQ ID NO:7);
ILPWKWPWWPWKKWK (SEQ ID NO:8);
PWKWPWWPWRR (SEQ ID NO:9);
ILPWKWPWRR (SEQ ID NO:10);
ILPWKWPWWPWWPWRR (SEQ ID NO:11);
ILPWKWPWWPWWKKPWRR (SEQ ID NO:12);
ILPWICPWRPSKAN (SEQ ID NO:13);
IVPWKWTLWPWRR (SEQ ID NO:14);
TLPCLWPWWPWSI (SEQ ID NO:15);
ILKKWPWWPWKRR (SEQ ID NO:17);
ILKKWPWWPWKWKK (SEQ ID NO: 18);
ILPWKWPWYVRR (SEQ ID NO: 19);
IKWPWYVWL (SEQ ID NO: 20);
ILPWKWFFPPWPWRR (SEQ ID NO: 21);
ILPWKWPPWPPWPWRR (SEQ ID NO: 22);
ILKKWPWWRWRR (SEQ ID NO: 27);
ILKKFPFFPFRRK (SEQ ID NO: 28);
ILKKFPFFPFKKK (SEQ ID NO: 29);
ILKKWAWWPWRRK (SEQ ID NO: 30);
ILKKWPWWAWRRK (SEQ ID NO: 31);
ILKKWPWWPWKKK (SEQ ID NO: 32);
ILRRWPWWPWRRR (SEQ ID NO: 33);
WWKKWPWWPWRRK (SEQ ID NO: 34);
FFKKWPWWPWRRK (SEQ ID NO: 35);
FFKKFPFFPFRRK (SEQ ID NO: 36);
FFKKFPFFPFKKK (SEQ ID NO: 37);
ILKKWPWWPWWPWRRK (SEQ ID NO: 38);
ILKKWPWWPWRWWRR (SEQ ID NO: 39);
ILKKWPWWPWRRWWK (SEQ ID NO: 40);
ILKKWPWWPWPPRRK (SEQ ID NO: 41);
ILKKWPWWPWPPFFRRK (SEQ ID NO: 42);
The invention also provides a method of inhibiting the growth of a
bacterium (or bacteria) by contacting the bacterium with an inhibiting
effective amount of one or more of the peptides of the invention, used
simultaneously or sequentially. If desired, the peptide(s) can be used in
combination with an antibiotic, simultaneously or sequentially. Such
combination therapy may prove beneficial by a synergy occurring between
the peptide and the antibiotic.
The invention also provides a method for inhibiting an endotoxemia or
sepsis-associated disorder in a subject (e.g., a mammal such as a human)
that has, or is at risk of having, such a disorder; the method entails
administering to the subject a therapeutically effective amount of a
peptide of the invention.
In addition, the invention provides a method for producing a cationic
peptide variant having antimicrobial activity. This method entails:
identifying the amino acid sequence of a reference cationic peptide having
antimicrobial activity; producing an expression library encoding cationic
peptide variants of the reference peptide (where a plurality of the
variants each contain at least one substitution of an amino acid of the
identified amino acid sequence); expressing the library in a plurality of
host cells (thereby creating a plurality of clones); and isolating a clone
that produces a cationic peptide variant having antimicrobial activity.
The invention provides several advantages. The peptides of the invention
are compact and tend to have a unique polyproline type II extended helix
structure that permits them to span the membrane with relatively few amino
acids. The best peptides have very broad spectrum activity against
antibiotic resistant bacteria, combined with activity against the
medically important fungus, Candida albicans. These peptides also often
possess the ability to work synergistically with antibiotics; in addition,
they often possess anti-endotoxin activity. The peptides can be produced
efficiently by recombinant DNA and protein chemical means. The invention
also provides methods that permit both rationally designed and semi-random
mutants of core structures (e.g., reference proteins) to be tested to
permit variants within the formulas described herein to be produced and
tested.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows CD spectra for CP-11 and indolicidin.
FIG. 2A shows the structure of indolicidin.
FIG. 2B shows a transverse view of the indolicidin structure.
FIG. 3 shows a microbial killing curve for CP-11.
FIG. 4 shows the ability of antimicrobial peptides to permeabilize the
outer membrane of bacteria.
FIG. 5 shows the ability of antimicrobial peptides to permeabilize the
inner membrane of bacteria.
FIG. 6 shows the voltage/current relationship for indolicidin.
FIG. 7 shows the voltage sign dependence of indolicidin.
FIG. 8 shows single channel conductances produced by indolicidin in planar
lipid bilayers.
FIG. 9 shows the effects of peptides on E. coli 0111:B4 LPS-induced TNF.
FIG. 10 shows the effect of peptides on P. aeruginosa LPS-induced TNF.
FIG. 11 shows the effect of cationic peptides on LPS released by P.
aeruginosa.
FIG. 12 shows the effect of cationic peptides on LPS released by Bort E.
coli.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a novel class of cationic peptides (i.e.,
polypeptides) that have antimicrobial activity. These peptides are useful
for inhibiting microbial infection or growth, as well reducing the effects
of endotoxemia. The peptides can be used, for example, as preservatives in
foods or cosmetics. Many of the peptides of the invention are synergistic
with conventional antibiotics and can be used as an adjunct therapy. In
addition, such peptides are useful as antifungal agents, antitumor agents,
and/or antiviral agents.
The term "antimicrobial" as used herein means that the peptide destroys, or
inhibits or prevents the growth or proliferation of, a microbe (e.g., a
bacterium, fungus, and/or virus). Likewise, the term "antiviral" as used
herein means that a peptide destroys, or inhibits or prevents the growth
or proliferation of, a virus or a virus-infected cell. The term
"anti-tumor" as used herein means that a peptide prevents, inhibits the
growth of, or destroys, a tumor cell(s). Similarly, the term "antifungal"
means that a peptide prevents, destroys, or inhibits the growth of a
fungus.
As used herein, the term "cationic peptide" refers to a chain of amino
acids that is 5 to 50 (preferably 9 to 25) amino acids in length. A
peptide is "cationic" if it has a pKa greater than 9.0. Typically, at
least four of the amino acid residues of the cationic peptide are
positively charged residues, e.g., lysine and arginine. "Positively
charged" refers to the side chain of an amino acid residue that has a net
positive charge at pH 7.0.
As is described below, Applicants have devised formulas that encompass the
isolated peptides of the invention. These peptides are represented by the
amino acid sequences:
X.sub.1 X.sub.1 PX.sub.2 X.sub.3 X.sub.2 P(X.sub.2 X.sub.2 P).sub.n
X.sub.2 X.sub.3 (X.sub.5).sub.o ; (SEQ ID NO: 23)
X.sub.1 X.sub.1 PX.sub.2 X.sub.3 X.sub.4 (X.sub.5).sub.r PX.sub.2
X.sub.3 X.sub.3; (SEQ ID NO: 24)
X.sub.1 X.sub.1 X.sub.3 (PW).sub.u X.sub.3 X.sub.2 X.sub.5 X.sub.2
X.sub.2 X.sub.5 X.sub.2 (X.sub.5).sub.o ; and (SEQ ID NO: 25)
X.sub.1 X.sub.1 X.sub.3 X.sub.3 X.sub.2 P(X.sub.2 X.sub.2 P).sub.n
X.sub.2 (X.sub.5).sub.m ; (SEQ ID NO: 26)
wherein:
m is 1 to 5;
n is 1 or 2;
o is 2 to 5;
r is 0 to 8;
u is 0 or 1;
X.sub.1 is Isoleucine, Leucine, Valine, Phenylalanine, Tyrosine, Tryptophan
or Methionine;
X.sub.2 represents Tryptophan or Phenylalanine
X.sub.3 represents Arginine or Lysine;
X.sub.4 represents Tryptophan or Lysine; and
X.sub.5 represents Phenylalanine, Tryptophan, Arginine, Lysine, or Proline.
The term "isolated" as used herein refers to a peptide that is
substantially free of other proteins, lipids, and nucleic acids (e.g.,
cellular components with which an in vivo-produced peptide would naturally
be associated). Preferably, the peptide is at least 70%, 80%, or most
preferably 90% pure by weight.
The invention also includes analogs, derivatives, conservative variations,
and cationic peptide variants of the enumerated polypeptides, provided
that the analog, derivative, conservative variation, or variant has a
detectable antimicrobial activity. It is not necessary that the analog,
derivative, variation, or variant have activity identical to the activity
of the peptide from which the analog, derivative, conservative variation,
or variant is derived.
A cationic peptide "variant" is an antimicrobial peptide that is an altered
form of a referenced antimicrobial cationic peptide. For example, the term
"variant" includes an antimicrobial cationic peptide produced by the
method disclosed herein in which at least one amino acid of a reference
peptide is substituted in an expression library. The term "reference"
peptide means any of the antimicrobial cationic peptides of the invention
(e.g., as defined in the above formulas), from which a variant,
derivative, analog, or conservative variation is derived. Included within
the term "derivative" is a hybrid peptide that includes at least a portion
of each of two antimicrobial cationic peptides (e.g., 30-80% of each of
two antimicrobial cationic peptides). Also included are peptides in which
one or more amino acids are deleted from the sequence of a peptide
enumerated herein, provided that the derivative has antimicrobial
activity. For example, amino or carboxy terminal amino acids that are not
be required for antimicrobial activity of a peptide can be removed.
Likewise, additional derivatives can be produced by adding one or a few
(e.g., less than 5) amino acids to an antimicrobial peptide without
completely inhibiting the antimicrobial activity of the peptide. In
addition, C-terminal derivatives, e.g., C-terminal methyl esters, can be
produced and are encompassed by the invention.
The invention also includes peptides that are conservative variations of
those peptides exemplified herein. The term "conservative variation" as
used herein denotes a polypeptide in which at least one amino acid is
replaced by another, biologically similar residue. Examples of
conservative variations include the substitution of one hydrophobic
residue, such as isoleucine, valine, leucine, alanine, cysteine, glycine,
phenylalanine, proline, tryptophan, tyrosine, norleucine or methionine for
another, or the substitution of one polar residue for another, such as the
substitution of arginine for lysine, glutamic for aspartic acid, or
glutamine for asparagine, and the like. Neutral hydrophilic amino acids
that can be substituted for one another include asparagine, glutamine,
serine and threonine. The term "conservative variation" also encompasses a
peptide having a substituted amino acid in place of an unsubstituted
parent amino acid; preferably, antibodies raised to the substituted
polypeptide also specifically bind the unsubstituted polypeptide.
The activity of the peptides of the invention can be determined using
conventional methods known to those of skill in the art, such as in a
"minimal inhibitory concentration (MIC)" assay described herein, whereby
the lowest concentration at which no change in OD is observed for a given
period of time is recorded as the MIC. Alternatively, a "fractional
inhibitory concentration (FIC)" assay can be used to measure synergy
between the peptides of the invention, or the peptides in combination with
known antibiotics. FICs can be performed by checkerboard titrations of
peptides in one dimension of a microtiter plate, and of antibiotics in the
other dimension, for example. The FIC is a function of the impact of one
antibiotic on the MIC of the other and vice versa. A FIC of 1 indicates
that the influence of the compounds is additive and a FIC of less than 1
indicates that the compounds act synergistically.
Peptides of the invention can be synthesized by commonly used methods such
as those that include t-BOC or FMOC protection of alpha-amino groups. Both
methods involve stepwise synthesis in which a single amino acid is added
at each step starting from the C terminus of the peptide (See, Coligan, et
al., Current Protocols in Immunology, Wiley Interscience, 1991, Unit 9).
Peptides of the invention can also be synthesized by the well known solid
phase peptide synthesis methods such as those described by Merrifield, J.
Am. Chem. Soc., 85:2149, 1962) and Stewart and Young, Solid Phase Peptides
Synthesis, Freeman, San Francisco, 1969, pp.27-62) using a
copoly(styrene-divinylbenzene) containing 0.1-1.0 mMol amines/g polymer.
On completion of chemical synthesis, the peptides can be deprotected and
cleaved from the polymer by treatment with liquid HF-10% anisole for about
1/4-1 hours at 0.degree. C. After evaporation of the reagents, the
peptides are extracted from the polymer with a 1% acetic acid solution,
which is then lyophilized to yield the crude material. The peptides can be
purified by such techniques as gel filtration on Sephadex G-15 using 5%
acetic acid as a solvent. Lyophilization of appropriate fractions of the
column eluate yield homogeneous peptide, which can then be characterized
by standard techniques such as amino acid analysis, thin layer
chromatography, high performance liquid chromatography, ultraviolet
absorption spectroscopy, molar rotation, or measuring solubility. If
desired, the peptides can be quantitated by the solid phase Edman
degradation.
The invention also includes isolated nucleic acids (e.g., DNA, cDNA, or
RNA) encoding the peptides of the invention. Included are nucleic acids
that encode analogs, mutants, conservative variations, and variants of the
peptides described herein. The term "isolated" as used herein refers to a
nucleic acid that is substantially free of proteins, lipids, and other
nucleic acids with which an in vivo-produced nucleic acids naturally
associated. Preferably, the nucleic acid is at least 70%, 80%, or
preferably 90% pure by weight, and conventional methods for synthesizing
nucleic acids in vitro can be used in lieu of in vivo methods. As used
herein, "nucleic acid" refers to a polymer of deoxyribo-nucleotides or
ribonucleotides, in the form of a separate fragment or as a component of a
larger genetic construct (e.g., by operably linking a promoter to a
nucleic acid encoding a peptide of the invention). Numerous genetic
constructs (e.g., plasmids and other expression vectors) are known in the
art and can be used to produce the peptides of the invention in cell-free
systems or prokaryotic or eukaryotic (e.g., yeast, insect, or mammalian)
cells. By taking into account the degeneracy of the genetic code, one of
ordinary skill in the art can readily synthesize nucleic acids encoding
the polypeptides of the invention. The nucleic acids of the invention can
readily be used in conventional molecular biology methods to produce the
peptides of the invention.
DNA encoding the cationic peptides of the invention can be inserted into an
"expression vector." The term "expression vector" refers to a genetic
construct such as a plasmid, virus or other vehicle known in the art that
can be engineered to contain a nucleic acid encoding a polypeptide of the
invention. Such expression vectors are preferably plasmids that contain a
promoter sequence that facilitates transcription of the inserted genetic
sequence in a host cell. The expression vector typically contains an
origin of replication, and a promoter, as well as genes that allow
phenotypic selection of the transformed cells (e.g., an antibiotic
resistance gene). Various promoters, including inducible and constitutive
promoters, can be utilized in the invention. Typically, the expression
vector contains a replicon site and control sequences that are derived
from a species compatible with the host cell.
Transformation or transfection of a host cell with a nucleic acid of the
invention can be carried out using conventional techniques well known to
those skilled in the art. For example, where the host cell is E. coli,
competent cells that are capable of DNA uptake can be prepared using the
CaCl.sub.2, MgCl.sub.2 or RbCl methods known in the art. Alternatively,
physical means, such as electroporation or microinjection can be used.
Electroporation allows transfer of a nucleic acid into a cell by high
voltage electric impulse. Additionally, nucleic acids can be introduced
into host cells by protoplast fusion, using methods well known in the art.
Suitable methods for transforming eukaryotic cells, such as
electroporation and lipofection, also are known.
"Host cells" encompassed by of the invention are any cells in which the
nucleic acids of the invention can be used to express the polypeptides of
the invention. The term also includes any progeny of a host cell.
Preferred host cells of the invention include E. coli, S. aureus and P.
aeruginosa.
Nucleic acids encoding the peptides of the invention can be isolated from a
cell (e.g., a cultured cell), or they can be produced in vitro. A DNA
sequence encoding a cationic peptide of interest can be obtained by: 1)
isolation of a double-stranded DNA sequence from genomic DNA; 2) chemical
manufacture of a nucleic acid such that it encodes the cationic peptide of
interest; or 3) in vitro synthesis of a double-stranded DNA sequence by
reverse transcription of mRNA isolated from a donor cell (i.e., to produce
cDNA). Among the standard procedures for isolating cDNA sequences of
interest is the formation of plasmid or phage containing cDNA libraries
that are derived from reverse transcription of mRNA in donor cells that
have a high level of genetic expression. When used in combination with
polymerase chain reaction technology, even rare gene products can be
cloned.
The invention also provides a method for inhibiting the growth of a
bacterium by contacting the bacterium with an inhibiting effective amount
of a peptide of the invention. The term "contacting" refers to exposing
the bacterium to the peptide so that the peptide can inhibit, kill, or
lyse bacteria. Preferably, the peptide is able to bind endotoxin (LPS), or
permeabilize the outer membrane of a gram-negative bacterium. Contacting
can occur in vitro, for example, by adding the peptide to a bacterial
culture to test for susceptibility of the bacteria to the peptide.
Alternatively, contacting can occur in vivo, for example by administering
the peptide to a subject afflicted with a bacterial disorder, such as
septic shock. "Inhibiting" or "inhibiting effective amount" refers to the
amount of peptide that is sufficient to cause a bacteriostatic or
bactericidal effect. Examples of bacteria that can be inhibited include E.
coli, P. aeruginosa, E. cloacae, S. typhimurium, and S. aureus. The method
for inhibiting the growth of bacteria can also include the contacting the
bacterium with the peptide in combination with one or more antibiotics.
A peptide(s) of the invention can be administered to any host, including a
human or non-human animal, in an amount effective to inhibit growth of a
bacterium, virus, or fungus. Thus, the peptides are useful as
antimicrobial agents, antiviral agents, and/or antifungal agents.
Any of a variety of art-known methods can be used to administer the peptide
to a subject. For example, the peptide of the invention can be
administered parenterally by injection or by gradual infusion over time.
The peptide can be administered intravenously, intraperitoneally,
intramuscularly, subcutaneously, intracavity, or transdermally. It may be
formulated into liposomes to reduce toxicity or increase bio availability.
Other preferred methods for delivery of the peptide include oral methods
that entail encapsulation of the peptide in microspheres or proteinoids,
aerosol delivery (e.g., to the lungs), or transdermal delivery (e.g., by
iontophoresis or transdermal electroporation). Other methods of
administration will be known to those skilled in the art.
Preparations for parenteral administration of a peptide of the invention
include sterile aqueous or non-aqueous solutions, suspensions, and
emulsions. Examples of non-aqueous solvents are propylene glycol,
polyethylene glycol, vegetable oils (e.g., olive oil), and injectable
organic esters such as ethyl oleate. Examples of aqueous carriers include
water, saline, and buffered media, alcoholic/aqueous solutions, and
emulsions or suspensions. Examples of parenteral vehicles include sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's, and fixed oils. Intravenous vehicles include fluid and
nutrient replenishers, electrolyte replenishers (such as those based on
Ringer's dextrose), and the like. Preservatives and other additives such
as, other antimicrobial, anti-oxidants, cheating agents, inert gases and
the like also can be included.
The invention provides a method for inhibiting an endotoxemia or septic
shock (sepsis)-associated disorder by administering a therapeutically
effective amount of a peptide of the invention to a subject who has, or is
at risk of having, such a disorder. The term "inhibiting" means preventing
or ameliorating a sign or symptoms of a disorder (e.g., endotoxemia).
Examples of disease signs that can be ameliorated include an increase in a
patient's blood level of TNF, fever, hypotension, neutropenia, leukopenia,
thrombocytopenia, disseminated intravascular coagulation, adult
respiratory distress syndrome, shock, and organ failure. Examples of
patients who can be treated in the invention include those at risk for, or
those suffering from, a toxemia, such as endotoxemia resulting from a
gram-negative bacterial infection, venom poisoning, or hepatic failure.
Other examples include patients infected with gram-positive bacteria,
virus, or fungus. In particular, the invention is useful for treating
patients who display signs of sepsis, or complain of symptoms of sepsis.
Examples of candidate patients include those suffering from infection by
E. coli, Hemophilus influenza B, Neisseria meningitides, staphylococci, or
pneumococci. Other patients at risk for sepsis include those suffering
from gunshot wounds, renal or hepatic failure, trauma, burns,
immuno-compromising infections (e.g., HIV infections), hematopoietic
neoplasias, multiple myeloma, Castleman's disease or cardiac myxoma. Those
skilled in the art of medicine can readily employ conventional criteria to
identify appropriate subjects for treatment in accordance with the
invention.
The term "therapeutically effective amount" as used herein for treatment of
a patient afflicted with a disease or disorder means an amount of cationic
peptide sufficient ameliorate a sign or symptom of the disease. For
example, a therapeutically effective amount can be measured as the amount
sufficient to decrease a subject's response to LPS or lessen any sign or
symptom of sepsis. Preferably, the subject is treated with an amount of
cationic peptide sufficient to reduce an LPS-induced increase in plasma
TNF levels by at least 50%, and more preferably 90% or 100%. Generally,
the optimal dosage of the peptide will depend upon the disorder and
factors such as the weight of the patient. Nonetheless, suitable dosages
can readily be determined by one skilled in the art. If desired, the
effectiveness of treatment typically can be measured by monitoring the
level of LPS or TNF in a patient. A decrease in serum LPS and TNF levels
generally is correlated with amelioration of the disorder. Typically, a
suitable dosage is 0.5 to 40 mg peptide/kg body weight, preferably 1 to 8
mg peptide/kg body weight.
If desired, a suitable therapy regime can combine administration of a
peptide(s) of the invention with an inhibitor of TNF, an antibiotic, or
both. For example, intervention in the role of TNF in sepsis, either
directly or indirectly, such as by use of an anti-TNF antibody and/or a
TNF antagonist, can prevent or ameliorate the signs of sepsis. An anti-TNF
antibody, such as a monoclonal antibody that specifically binds TNF (e.g.,
as described by Tracey, el al. Nature, 330:662, 1987) is particularly
preferred. The peptide(s), inhibitor(s), and/or antibiotic(s) can be
administered, preferably, simultaneously, but may also be administered
sequentially. Suitable antibiotics include aminoglycosides (e.g.,
gentamicin), beta-lactams (e.g., penicillins and cephalosporins),
quinolones (e.g., ciprofloxacin), and novobiocin. Generally, the
antibiotic is administered in a bactericidal amount. However, the peptide
provides for a method of increasing antibiotic activity by permeabilizing
the bacterial outer membrane and combinations involving peptide and a
sub-inhibitory amount (an amount lower than the bactericidal amount) of
antibiotic can be administered. Preferably, the cationic peptide and
antibiotic are administered within 48 hours of each other (preferably 2-8
hours, most preferably, simultaneously). A "bactericidal amount" of
antibiotic is an amount sufficient to achieve a bacteria-killing blood
concentration in the patient receiving the treatment. In accordance with
its conventional definition, an "antibiotic," as used herein, is a
chemical substance produced by a microorganism that, in dilute solutions,
inhibits the growth of, or kills, other microorganisms. Also encompassed
by this term are synthetic antibiotics (e.g., analogs) known in the art.
The peptides of the invention can be used, for example, as preservatives or
sterilants of materials susceptible to microbial or viral contamination.
For example, the peptides can be used as preservatives in processed foods
(e.g., to inhibit organisms such as Salmonella, Yersinia, and Shigella).
If desired, the peptides can be used in combination with antibacterial
food additives, such as lysozymes. The peptides of the invention also can
be used as a topical agent , for example, to inhibit Pseudomonas or
Streptococcus or kill odor-producing microbes (e.g., Micrococci). The
optimal amount of a cationic peptide of the invention for any given
application can be readily determined by one of skill in the art.
The invention also provides a method for producing an antimicrobial
cationic peptide variant. This method entails identifying the amino acid
sequence of a cationic peptide having antimicrobial activity (i.e., a
reference peptide), producing an expression library encoding peptide
variants of the reference peptide, where each variant contains at least
one substitution (e.g., a random substitutions) of an amino acid(s)
identified in the reference peptide; expressing the library in a plurality
of host cells, thereby producing a plurality of clones; and isolating a
clone that produces a peptide variant having antimicrobial activity. The
invention also includes a cationic peptide variant produced by this
method.
In this method, randomized nucleotide substitutions can be introduced into
DNA encoding a reference cationic peptide having antimicrobial activity.
Peptides having amino acid substitutions can thus be produced. For
example, the example provided herein exemplifies randomization of a known
antimicrobial peptide in order to produce variants having antimicrobial
activity.
While any method of site directed mutagenesis can be used to produce
cationic peptide variants, the preferred method utilizes DNA synthesis
with a set of nucleotides in which each nucleotide stock, e.g., A,T,C, and
G, contains a low percentage of the other nucleotides. For example, the
adenosine stock contains about 0.5 to 5% of each of thymine, cytosine, and
guanine. It is understood that the greater the level of "contaminating"
nucleotides (i.e., T, C, and G in this example), the greater the number of
nucleotide substitutions and corresponding amino acid substitutions per
peptide.
Polymerase chain reaction (PCR) methods can be used to randomize specific
regions of an oligonucleotide encoding a cationic peptide of the
invention. PCR methods are well known in the art and are further
illustrated in the Examples disclosed herein. The products of the PCR
reactions can be pooled and gel purified prior to ligation into an
expression vector to form a library. By expressing the library in a
plurality of host cells (e.g., a strain of bacteria), a plurality of
clones is produced. A clone that produces a cationic peptide variant
having antimicrobial activity can readily be isolated by using a
convention assay (e.g., a MIC assay as described herein and as known to
those of skill in the art).
PCR primers used to amplify a nucleic acid encoding a variant cationic
peptide typically are complementary to regions of the expression vector
flanking the sequence encoding the variant cationic peptide.
Any nucleic acid sample, in purified or nonpurified form, can be used as a
nucleic acid template for DNA synthesis, provided it contains a nucleic
acid encoding the reference antimicrobial cationic peptide. Thus, the
process can employ single stranded or double stranded DNA or RNA, such as
messenger RNA. When RNA is used as a template, enzymes and conditions for
reverse transcribing the RNA template to DNA are utilized. If desired, a
DNA-RNA hybrid that contains one strand of each nucleic acid can be
utilized. If desired, a mixture of nucleic acids can be employed, or the
nucleic acids produced in a previous amplification reaction as described
herein can be used with the same or different primers. It is not necessary
that the nucleic acid to be amplified be present in a pure form; it can
constitute a minor fraction of a complex mixture.
Where the nucleic acid encoding the reference protein is double-stranded,
it is necessary to separate the strands of the nucleic acid in order to
provide a nucleic acid template. This strand separation can be
accomplished using any of various art-known denaturing conditions (e.g.,
physical, chemical, or enzymatic means). An example of a physical method
of separating nucleic acid strands is heating the nucleic acid to about
80.degree. to 105.degree. C. for 1 to 10 minutes, followed by rapid
cooling. Alternatively, strand separation can also be induced with an
enzyme from the class of enzymes known as helicase or by the enzyme RecA,
which has helicase activity. Reaction conditions suitable for strand
separation of nucleic acids with helicases or RecA are known in the art
(Kuhn Hoffmann-Berling CSH-Quantitative Biology, 43:63, 1978 and Radding,
Ann. Rev. Genetics, 16:405-437, 1982).
Where the nucleic acid containing the sequence to be amplified is single
stranded, its complement can be synthesized by using one or two
oligonucleotide primers. If a single primer is utilized, a primer
extension product can be synthesized in the presence of an agent for
polymerization and the four common nucleotide triphosphates.
When complementary strands of nucleic acid or acids are separated,
regardless of whether the nucleic acid was originally double or single
stranded, the separated strands can be used as templates for the synthesis
of additional nucleic acid strands. Typically, DNA synthesis occurs in a
buffered aqueous solution, preferably at a pH of 7-9, most preferably
about 8. Preferably, a molar excess (for genomic nucleic acid, usually
about 10.sup.8 :1 primer:template) of the two oligonucleotide primers is
added to the buffer containing the separated template strands. In
practice, the amount of primer added will generally be in molar excess
over the amount of complementary strand (template) when the sequence to be
amplified is contained in a mixture of complicated long-chain nucleic acid
strands.
Typically, the deoxyribonucleotide triphosphates dATP, dCTP, dGTP, and dTTP
are added to the synthesis mixture, either separately or together with the
primers, in adequate amounts and the resulting solution is heated to about
90.degree.-100.degree. C. from about 1 to 10 minutes, preferably from 1 to
4 minutes. If desired, nucleotide analogs can be used in lieu of, in
addition to, the common nucleotides. After this heating period, the
solution typically is allowed to cool to a temperature that is optimal for
primer hybridization (approximately 42.degree. C.). To the cooled mixture
is added an appropriate agent for effecting the primer extension reaction
(called herein "agent for polymerization"), and the synthesis reaction is
allowed to proceed under conditions known in the art. The agent for
polymerization (e.g., a thermostable polymerase) may also be added
together with the other reagents. This synthesis (or amplification)
reaction can occur at any temperature at which agent for polymerization
functions.
The agent for polymerization can be any compound (e.g., an enzyme) or
system that functions to synthesize primer extension products. Suitable
enzymes for this purpose include, for example, E. coli DNA polymerase I,
Klenow fragment of E. coli DNA polymerase I, T4 DNA polymerase, polymerase
muteins, reverse transcriptase, and other enzymes, especially heat-stable
polymerases. The double-stranded molecule that results from DNA synthesis
is then denatured, and the above-described synthesis process can be
repeated on the resulting single-stranded nucleic acids. Additional agent
for polymerization, nucleotides, and primers can be added if desired. The
denaturing and DNA synthesis steps can be repeated if desired (as in
conventional PCR methods).
Nucleic acids of the invention can be evaluated, detected, cloned,
sequenced, and the like, either in solution or after binding to a solid
support, by any method known in the art (e.g., PCR, oligomer restriction
(Saiki, et al., Bio/Technology, 3:1008-1012, 1985), allele-specific
oligonucleotide (ASO) probe analysis (Conner, et al., Proc. Natl. Acad.
Sci. USA, 80:278, 1983), oligonucleotide ligation assays (OLAs)
(Landegren, et al., Science, 241:1077, 1988), fluorescent in situ
hybridization (FISH), and the like).
In the present invention, a nucleic acid encoding a cationic peptide or
peptide variants can be inserted into a recombinant "expression vector."
The term "expression vector" refers to a plasmid, virus or other vehicle
known in the art that can be manipulated by insertion or incorporation of
a nucleic acid encoding a cationic peptide or variant. Typically,
expression vectors are plasmids that contain a promoter for directing
transcription of the inserted genetic sequence.
If desired, the expression vector can encode a "carrier peptide," which
typically is produced as a fusion with the amino terminus of the peptide
variant. Preferably, the carrier peptide is sufficiently anionic such that
the positive charge associated with the cationic peptide is overcome and
the resulting fusion peptide has a net charge that is neutral or negative.
The anionic carrier peptide can correspond in sequence to a
naturally-occurring protein or can be entirely artificial in design.
Functionally, the carrier peptide may help stabilize the cationic peptide
and protect it from proteases, although the carrier peptide need not be
shown to serve such a purpose. Similarly, the carrier peptide may
facilitate transport of the fusion peptide. Examples of carrier peptide
that can be utilized include anionic pre-pro peptides and anionic outer
membrane peptides. Preferred carrier peptides include, but are not limited
to, glutathione-S-transferase (GST) (Smith et al. Proc. Natl. Acad. Sci.
USA, 83:8703, 1986), protein A of Staphylococcus aureus (Nilsson, et al.,
EMBO, 4:1075, 1985; pRIT5 (Pharmacia)), two synthetic IgG-binding domains
(ZZ) of protein A (Lowenadler, et al., Gene, 58:87, 1987) and outer
membrane protein F of Pseudomonas aeruginosa (Duchene, et al., J.
Bacteriol, 170:155, 1988). The invention is not limited to the use of
these peptides as carriers; others suitable carrier peptides are known to
those skilled in the art. Alternatively, the carrier peptide can be
omitted altogether.
Any of various art-known methods for protein purification can be used to
isolate the peptides of the invention. For example, preparative
chromatographic separations and immunological separations (such as those
employing monoclonal or polyclonal antibodies) can be used. Carrier
peptides can facilitate isolation of fusion proteins that include the
peptides of the invention. For example, glutathione-S-transferase (GST)
allows purification with a glutathione agarose affinity column. When
either Protein A or the ZZ domain from Staphylococcus aureus is used as
the carrier protein, purification can be accomplished in a single step
using an IgG-sepharose affinity column. The pOprF-peptide, which is the
N-terminal half of the P. aeruginosa outer membrane protein F, can readily
be purified because it is the prominent protein species in outer membrane
preparations. If desired, the fusion peptides can be isolated by using
reagents that are specifically reactive with (e.g., specifically bind) the
cationic peptide of the fusion peptide. For example, monoclonal or
polyclonal antibodies that specifically bind the cationic peptide can be
used in conventional purification methods. Techniques for producing such
antibodies are well known in the art.
In practicing the invention, it may be advantageous to include a "spacer
DNA sequence" in the expression vectors. As used herein, "spacer DNA
sequence" refers to any coding sequence located between the sequence
encoding the carrier peptide and the sequence encoding the cationic
peptide. While not wanting to be bound to a particular theory, it is
believed that the spacer DNA sequence, when translated, can create a
"hinge-like" region that allows the negatively charged residues of the
anionic carrier peptide and the positively charged residues of the subject
cationic peptide to interact, thereby inhibiting positive charge effects.
If desired, the spacer DNA sequence can encode a protein recognition site
for cleavage of the carrier peptide from the fusion peptide. Examples of
such spacer DNA sequences include, but are not limited to, protease
cleavage sequences, such as that for Factor Xa protease, the methionine,
tryptophan and glutamic acid codon sequences, and the pre-pro defensin
sequence. Factor Xa is used for proteolytic cleavage at the Factor Xa
protease cleavage sequence, while chemical cleavage by cyanogen bromide
treatment releases the peptide at the methionine or related codons. In
addition, the fused product can be cleaved by insertion of a codon for
tryptophan (cleavable by o-iodosobenzoic acid) or glutamic acid (cleavable
by Staphylococcus protease). Insertion of such spacer DNA sequences is not
a requirement for the production of functional cationic peptides, such
sequences can enhance the stability of the fusion peptide. The pre-pro
defensin sequence is negatively charged; accordingly, it is envisioned
within the invention that other DNA sequences encoding negatively charged
peptides also can be used as spacer DNA sequences to stabilize the fusion
peptide.
The following examples are intended to illustrate but not limit the
invention. While they are typical of those that might be used, other
procedures known to those skilled in the art may alternatively be used.
EXAMPLES
Example I
Synthesis of Cationic Peptides
Cationic peptides of the invention can be synthesized according to
conventional protocols for protein synthesis (Merrifield, J. Am. Chem.
Soc., 85:2149, 1962; Stewart and Young, Solid Phase Peptide Synthesis,
Freeman, San Francisco, 1969, pp 27-62) . Generally, the resulting
peptides are substantially pure, i.e., at least 70% pure. Typically, the
resulting peptides are at least 80% pure, and more typically at least 99%
pure. For example, the cationic peptides, termed CP-11 and CP-13 were
synthesized by standard protein chemical procedures, and were at least 99%
pure. In the examples provided below, indolicidin (an art-known
antimicrobial agent) is used as a control. Indolicidin also was
synthesized according to conventional methods, and it was at least 99%
pure. The peptide CP-12 was approximately 80% pure, most likely because
its sequence contained a larger number of positively charges residues in
close proximity to tryptophans. Peptides CP-IA and CP-AA were synthesized
by employing FMOC chemistry. Amino terminal modifications were made to
peptides CP-11 and indolicidin; each was synthesized with the N-terminal
amino acid in the D-form, thereby producing CP-11D and indolicidin-D. In
addition, CP-11, CP-11D, indolicidin, and indolicidin-D each was
methyl-esterified at the C-terminus, thereby producing CP-11C, CP-11DC,
indolicidin-C, and indolicidin-DC, respectively. Also, CP-11 was
synthesized to contain an amidated C-terminus, thereby producing CP-11N.
Methods for producing peptides having an N-terminal amino acid, a
methyl-esterified C-terminus, or an amidated C-terminus are well known in
the art (Stewart and Young, supra; Kadaba, Synthesis, pp 628-631, 1992).
The primary amino acid sequences of indolicidin, CP-11, CP-12, and CP-13
are shown in Table 1. For comparison, two peptides that lack significant
antimicrobial activity, CP-IA and CP-AA, also were synthesized. CP-IA is
expected to act synergistically with antibiotics as an antimicrobial.
TABLE 1
Synthetic Cationic Peptides
Peptide Amino acid sequence
Indolicidin ILPWKWPWWPWRR (SEQ ID NO: 16)
CP-11 ILKKWPWWPWRRK (SEQ ID NO: 1)
CP-12 ILKPWKWPWWPWRRKK (SEQ ID NO: 2)
CP-13 ILKKWPWWPWKWKK (SEQ ID NO: 18)
CP-IA ILPWKWPWYVRR (SEQ ID NO: 19)
CP-AA IKWPWYVWL (SEQ ID NO: 20)
CP-16 ILKKFPFFPFRRK (SEQ ID NO: 28);
CP-24 FFKKFPFFPFRRK (SEQ ID NO: 36);
Example II
Circular Dichroism Analysis
By combining an analysis of peptide structure with peptide function (e.g.,
antimicrobial activity), Applicants have devised a formula that defines
the antimicrobial peptides of the invention (see below). Using Applicant's
formula, a person of ordinary skill in the art can readily produce an
antimicrobial peptide, such as a peptide specified herein, as well as
those peptides that are encompassed by the formula, but not specified
herein.
Structural analysis of indolicidin (a control) and CP-11 was performed
using conventional circular dichroism (CD) analysis and a J-720
spectropolarimeter (Jasco, Japan). Suitable methods for CD have been
described (see Haschmeyer and Haschmeyer, Proteins, A Guide to Study by
Physical and Chemical Methods, Wiley-Interscience, pp 237-253; 1973). In
this case, CD spectra were measured in 10 mM sodium phosphate buffer (pH
7.0), and in the presence or absence of
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
(POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) (7:3)
liposomes. Multilamellar liposomes were prepared by standard procedures
(see, e.g., Mayer et al., Biochim. Biophys. Acta 817 (1985) 193-196) and
extruded using an extruder device (Lipex Biomembranes, Vancouver, Canada)
to produce unilamellar vesicles. The concentration of peptide and
liposomes was 50 .mu.M and 2 mM, respectively.
In the absence of liposomes, indolicidin and CP-11 exhibited CD spectra
that were characteristic of an unordered structure (FIG. 1). In contrast,
in the presence of liposomes (thereby mimicking the peptide's structure
upon interaction with a cell membrane), the spectrum of indolicidin was
characteristic of that produced by a poly-L-proline II extended helix
structure. The spectrum was characterized by a minimum at 202 nm, a
maximum at 226 nm, and a smaller minimum at 235 nm. This spectrum was only
induced in the presence of negatively charges liposomes (i.e., such
structures were not induced in the presence of POPC liposomes alone). This
observation was not expected, because the low proline content of
indolicidin would have been expected to preclude such a structure.
Applicants' discovery that indolicidin formed a poly-L-proline II helix
was critical in devising a formula for the design of cationic peptides.
Using circular dichroism, CP-11 produced a spectra (FIG. 1), which,
although it had maximum and minimum at the same wavelengths as
indolicidin, was distinct from indolicidin in that the minima at 202 and
235 were reduced, and the maximum at 226 was increased in magnitude. Two
peptides that lack significant antimicrobial activity, CP-IA and CP-AA
(Table 1), also were analyzed by circular dichroism. Structural analysis
of the non-antimicrobial peptides CP-IA and CP-AA revealed that certain
motifs contribute significantly to the lipid-induced structure of the
antimicrobial peptides. Both CP-IA and CP-AA remained unordered both in
the absence of liposomes and in the presence of liposomes that were able
to induce structure to the antimicrobial peptides (indolicidin and CP-11).
Peptide IA differs from indolicidin in that it contains Phe.sup.9 and
Val.sup.10 in lieu of Trp.sup.9, Pro.sup.10, and Trp.sup.11 of
indolicidin. Both CP-IA and CP-AA have significantly lower antimicrobial
activities than does indolicidin (see below). Based on this structural
analysis, it is expected that a poly-L-proline II helix structure
contributes to antimicrobial activity.
Example III
Computer-based Modeling of Antimicrobial Peptides
Using InsightII (Biosym. Inc., San Diego, Calif.) molecular modeling
software, indolicidin was modeled using the co-ordinates for a
poly-L-proline II helix. The resulting structure is shown in FIG. 2A.
These data show that the peptide in this form is approximately 40 .ANG. in
length. Also, Trp.sup.6, Trp.sup.8, Trp.sup.9 and Trp.sup.11 are all
aligned in the same plane (FIG. 2B), as opposed to Trp.sup.4, which is
aligned in the opposite plane. Using CP-11 and the co-ordinates for a
poly-L-proline II helix, computer modeling showed that all of the
tryptophans of CP-11 were aligned in a single plane. Because CP-11 is a
more potent antimicrobial peptide than is indolicidin (see below), this
structural analysis combined with the functional analysis, suggests that
formation of an amphipathic structure, as in CP-11 is desirable.
Example IV
Assay for Antimicrobial Activity
Any of the art-known methods for measuring antimicrobial activity of
peptides can be used in characterizing the peptides of the invention. In
these examples, conventional methods were used to determine the minimal
inhibitory concentration (MIC) of a peptide in a liquid medium (Amsterdam.
Antibiotics in Laboratory Medicine, Williams and Wilkins, Baltimore (1991)
72-78). Briefly, overnight cultures of the test organisms (described
below) were diluted to produce an inoculum containing approximately
10.sup.4 to 10.sup.5 organisms. The inoculum size was confirmed by
counting the number of organisms on plates that were inoculated at the
same time as the liquid cultures.
The concentration of peptide was measured by assaying for free amino groups
using a dinitrophenylation assay, with polymyxin B serving as a standard.
Doubling dilutions of peptide were performed (32 .mu.g/ml-0.25 .mu.g/ml)
in 100 .mu.l volumes in a 96-well mictrotiter plate, and all wells were
subsequently inoculated with the diluted culture of the test organism.
After 24 hours of incubation at 37.degree. C., the assay was read. The MIC
value is the concentration of peptide at which the growth of the test
organism was reduced by 50%. The number of cells in the culture inoculum
was calculated from the plate count after incubating the plate for 24
hours at 37.degree. C. The medium used for cell culture and dilution in
this assay was Luria broth (LB). Each peptide was tested three times
against each organism, and the MIC values presented below are the mean
values of the three experiments. The test organisms used in the MIC assays
are listed in Table 2.
TABLE 2
Test strains used for MIC determination
Strain
Number Organism Source
UB1005 E. coli D. Clark
14028S Salmonella typhimurium Defensin sensitive (F. Heffron)
C621 Staphylococcus epidermidis Clinical isolate (D. Speert)
RN4220 Staphylococcus aureus ATCC 25293
C627 Candida albicans UBC Microbiology department
collection
K799 Pseudomonas aeruginosa W. Zimmerman
See Piers et al., Antimicrob. Agents. Chemother, 38:2311-2316, 1994 for a
citation to the Gram negative bacterial strains.
MIC values for each of the peptides are shown in Table 3. These data show
that the cationic peptide CP-11 had greater antimicrobial activity against
gram-negative bacteria and the yeast Candida albicans than did
indolicidin. Similarly, CP-13 displayed greater antimicrobial activity
against gram-negative bacteria than did indolicidin. The peptide CP-16,
which had Phenylalanine substituted for Tryptophan but otherwise had the
same sequence as CP-13, was equivalent to this peptide in activity against
S. aureus, showing that the peptides of the invention did not need to be
Tryptophan-rich. A further substitution of the first two residues in CP-24
for the hydrophobic amino acid phenylalanine led to a peptide that was
superior to indolicidin and better than CP-13 against S. aureus.
MIC values also were determined for various derivatives of indolicidin,
CP-11, and CP-13. As described above the C-terminus of indolicidin and
CP-11 was chemically converted to a methyl-ester, thereby producing
indolicidin-C and CP-11IC. The amidated derivative of CP-11, CP-11N, was
made by FMOC chemistry. In the case of indolicidin, methyl esterification
reduced the MIC for all organisms except P. aeruginosa and C. albicans. In
the case of CP-11, the methyl esterification reduced the MIC values for
all organisms except S. aureus SAP0017 and C. albicans. CP-11C produced
MIC values 8 fold lower than indolicidin for the gram-negative organisms
and C. albicans. CP-11 and CP-11C are particularly valuable for their
antimicrobial activity against P. aeruginosa. These results are attributed
to the increase in positive charge and orientation of the hydrophobic
residues of CP-11 and its derivatives. The peptide having the most potent
antimicrobial activity, CP-11C, exhibited MIC values of 1-8 .mu.g/ml for
all of the organisms tested.
TABLE 3
MIC values for synthetic peptides
MIC .mu.g/ml
P. aeru- S. typhi- S. epider- C. albi-
E. coli ginosa murium S. aureus midis cans
Indolicidin 16 >32 8 8 8 >32
CP-11 4 16 2 16 2 8
CP-13 8 >32 4 >32 4 >32
CP-16 16 64 16 16
CP-24 8 32 8 8
CP-IA >32 >32 >32 >32 >32 >32
CP-AA >32 >32 >32 >32 >32 >32
Indolici- 4 >32 2 4 1 >32
din-C
CP-11C 2 8 1 8 1 8
Indolici- 8 >32 4 4 2 >32
din-D
CP-11D 4 4 2 4 2 8
Indolici- 4 >32 4 8 2 >32
din-DC
CP-11DC 4 4 2 4 2 8
CP-11N 1 >32 8
Indolicidin, indolicidin C, and CP-11 also were synthesized such that the
N-terminal amino acid was in the D form, thereby producing indolicidin-D,
indolicidin-DC and CP-11C, respectively. The N-terminal modification of
CP-11 (creating CP-11D) decreased the MIC value for the peptide. The MIC
for CP-11DC was comparable to the MIC for CP-11D. Indolicidin-D exhibits
MIC values that are 1 to 2 dilution factors lower than indolicidin for the
gram-negative and gram-positive organisms tested (except P. aeruginosa).
In this example, indolicidin-DC had an approximately the same activity as
indolicidin-C.
Example V
Microbial Killing by CP-11
Killing curves were determined for CP-11 against E. coli, P. aeruginosa, S.
aureus and C. albicans (FIG. 3). This assay was performed in 10 mM HEPES
buffer, and viable counts were used to determine numbers of surviving
cells. In all cases, CP-11 (at 32 .mu.g/ml killed the test organisms
rapidly. Within 30 minutes of adding the peptide, the viable count of
gram-negative bacteria, gram-positive bacteria, and yeast decreased by at
least 3 log orders. These data show that CP-11 is a potent antimicrobial
peptide.
Example VI
Synergy Studies
In a variation of the MIC assay described above, the antimicrobial effects
of the peptides were measured in the presence of antibiotics or other
peptides. This assay showed that CP-11 and CP-13 were synergistic with
polymyxin B, cefepime, ceftazidime. CP-11 was also synergistic with
novobiocin and a cecropin/melittin (CEME) hybrid peptide (Piers, supra).
These experiments were performed by checkerboard titrations in which the
synergizing peptide was diluted in two fold steps in one direction in a
96-well microtitre tray and antibiotics or peptides were diluted at right
angles to the peptide. After addition of bacteria 10.sup.4 -10.sup.5 per
well, and overnight incubation at 37 .degree. C., a fractional inhibitory
concentration (FIC) was determined using an art known method (LeMan,
Antibiotics in Laboratory Medicine, 3rd ed., Williams and Wikins, pp
180-197, 1986). An FIC of 0.5 indicates synergism.
Example VIII
Binding of Antimicrobial Peptides to Purified Lipopolysaccharide
Peptides that bind lipopolysaccharide are thought to potentially function
as anti-endotoxins. The binding of various CP peptides to P. aeruginosa
H103 lipopolysaccharide (LPS) was measured in a dansyl polymyxin B (DPX)
displacement assay as described by Moore et al. (AAC, 26 (1986) 496-500).
Briefly, DPX exhibits enhanced fluorescence when it is bound to LPS.
Purified LPS was saturated with DPX by titrating LPS with samples of DPX
until maximum fluorescence was reached. Fluorescence was measured in a
Perkin-Elmer 650-10S fluorescence spectrophotometer and displacement of
DPX was measured as a decrease in fluorescence upon the addition of an
antimicrobial peptide.
Table 4 shows the percentage of DPX fluorescence remaining bound to LPS as
a function of peptide concentration. Each peptides I.sub.50 value, which
is the concentration at which 50% of the maximal DPX was displaced from
the LPS, is shown. Also shown is the I.sub.max value, which represents the
maximum displacement of LPS expressed as a percentage, where 100%
indicates displacement of all bound DPX.
TABLE 4
Binding of antimicrobial peptides to purified lipopolysaccharide
I.sub.50 of DPX displacement I.sub.max
Peptide .mu.M %
Indolicidin 8.5 62
Indolicidin-C 1.2 66
CP-11 4.3 75
CP-11C 3.1 78
As is shown above, CP-11C were more effective in displacing DPX from P.
aeruginosa LPS than the indolicidin. The relatively low I.sub.50
concentration of the CP-11 and CP-11C reflects the relatively high
affinity of these compounds for purified P. aeruginosa H103 LPS. The
I.sub.50 of Mg.sup.2+ ions in these experiments was approximately 620
.mu.M. These ions are the native divalent cations that normally bind LPS.
Therefore, all of the tested peptides were capable of binding surface LPS
by displacing such ions.
Example VIII
Membrane Permeabilization
The degree of outer membrane permeabilization of E. coli UB1005 by the
synthetic peptides was determined in a 1-N-phenylnapthylamine (NPN) uptake
assay (Loh et al. AAC, 26 (1984) 2311-2316). Briefly, NPN is a small (200
kD), hydrophobic molecule that when partitioned into the bacterial outer
membrane, exhibits an increase in fluorescence. Therefore, using a
fluorescence spectrophotometer, an increase in fluorescence in the
presence of a peptide indicates that the peptide can permeabilize the
bacterial outer membrane. Fluorescence is measured as a function of the
concentration of peptide, as shown in FIG. 4. This example shows that the
permeabilization of E. coli to NPN, by CP-11C was greater than that by
CP-11. Similarly, permeabilization by indolicidin-C was greater than that
by indolicidin. In addition CP-11 was substantially better able to
permeabilize the outer membrane of E. coli than was indolicidin.
Permeabilization of the E. coli inner membrane was measured using E. coli
ML-35, which is a lactose permease deficient strain that has constitutive
cytoplasmic .beta.-galactosidase activity (Lehrer et al., J. Clin. Invest.
84 (1989) 553-561). "Unmasking" of the .beta.-galactosidase activity by
due to permeabilizing of the inner membrane can be detected using the
substrate ONPG (o-nitrophenyl-.beta.-D-galactoside). Hydrolysis of ONPG
can be followed spectrophotometrically at 420 nm. FIG. 5 shows that CP-11
permeabilized the inner membrane of E. coli at peptide concentrations that
were 4 fold lower than the concentration of indolicidin required. Both of
the peptides permeabilized the membrane after little or no lag time. These
data show that, not only is the inner membrane the putative primary target
for CP-11, but also the peptide reaches this target rapidly. Therefore,
crossing of the outer membrane must also be a rapid process.
Example IX
Planar Lipid Bilayer Analysis
In order to gain insight into the mechanism by which these cationic
peptides permeabilize the cytoplasmic membrane of bacteria, the effects of
the peptide indolicidin on planar lipid bilayers were examined using the
standard technique of black lipid bilayer analysis (Benz and Hancock,
Biochim. Biophys. Acta 646 (1981) 298-308). Briefly, lipid bilayers (made
from 1.5% (w/v) phosphatidylcholine and phosphotidylserine (5:1 in
n-decane) were formed across a 0.2 mm.sup.2 hole separating two
compartments of a teflon chamber containing 1M KCl adjusted to pH 7.0 with
KH.sub.2 PO.sub.4. Calomel electrodes connected via a salt bridge
(Metrohm) were placed in each compartment, one connected to a voltage
source and the other connected to a Keithley-multimeter. The orientation
of the voltage was designated with respect to the addition of the peptide
to the cis-side with a trans-negative potential indicated by a minus sign.
To detect single depolarization events, one electrode was connected to a
current amplifier and chart recorder.
The current-voltage characteristics of indolicidin characterized by
macroscopic conductance experiments are shown in FIG. 6. The increase in
voltage had only a minor effect on the current produced by
permeabilization of the bilayer by indolicidin below -70 mV. At and above
-70 mV, there was a dramatic increase in current. The requirement by
indolicidin for a trans-membrane potential in excess of -70 mV makes the
action of the peptide against the cytoplasmic membrane voltage-dependent.
When the voltage was reduced from -80 mV back to 0 mV, the decrease was
linear (FIG. 6). This indicates that the threshold potential may only be
required to draw the peptide into the bilayer, and once this has occurred,
the channels remain relatively stable. Growing bacteria have a
trans-membrane potential in excess of -140 mV at neutral pH. The
requirement of a negative potential of 70 mV for activity was confirmed by
the reversal of the potential to +70 mV, resulting in a large reduction in
activity (FIG. 6).
The increase in membrane current caused by indolicidin was due, at least in
part, to the formation of single channels (FIG. 8). Single channel
conductances varied from 0.05-0.15 nS. However, channels of approximately
0.13 nS were repeatedly observed. CP-11 was also shown to form channels of
similar dimensions.
Example X
In vivo Efficacy of Antimicrobial Peptides
The in vivo efficacy of CP-11 and CP-11 CN was measured in a P. aeruginosa
infection model of immunosuppressed mice (Cryz et al. 39:3 (1983)
1067-1071). Mice were rendered leukopenic by a series of three
intraperitoneal (IP) injections of cyclophosphamide. CD-1 mice received
cyclophosphamide on Days 0, 2, and 4 at 150 .mu.g/g of mouse weight. On
Day 4, the mice were challenged with 100 live P. aeruginosa M2 bacteria,
and test groups received cationic peptide (8 mg/kg) in 100 .mu.l dH.sub.2
O at thirty minutes after injection of bacteria. In one experiment
(summarized in Table 6), mice received an additional cationic peptide
injection at 17 hours after bacterial challenge. The results of these
experiments are summarized in Tables 5 and 6.
TABLE 5
Influence of a single dose of cationic peptide on survival of
neutropenic mice challenged IP with P. aeruginosa strain M2
% Survival
Therapy 17 hr 24 hr 41 hr 48 hr 65 hr
No peptide 100 50 0 0 0
(n = 6)
CP-11CN 100 66.7 33.3 33.3 16.7
(n = 6)
CP-11 100 100 33.3 22.2 11.1
TABLE 6
Influence of two doses of cationic peptide on survival of neutropenic
mice challenged IP with P. aeruginosa
% Survival
Therapy 17 hr 24 hr 41 hr 48 hr 65 hr 72 hr
No peptide 100 86 14.3 0 0 0
(n = 7)
CP-11 100 100 37.5 25 25 25
(n = 8)
This example shows that CP-11 and CP-11CN each inhibit the fatal effects of
infection with P. aeruginosa. In addition, these data show that multiple
doses of the antimicrobial peptide prolong survival.
Example XI
Effect of Peptides on Red Blood Cells
This example shows that CP-11, CP-11C, and CP-13 are less toxic to human
cells than is indolicidin. Freshly collected human blood was mixed with
heparin and centrifuged to remove the buffy coat. The resulting
erythrocytes were washed three times in 0.85% saline and stored at
4.degree. C. Serial dilutions of the peptides in saline were prepared in
round bottomed microtitre plates using 100 .mu.l volumes. Red blood cells
were diluted with saline to 1/25 of the packed volume of cells and 50
.mu.l of red blood cells were added to each well. Plates were incubated
while rocking at 37.degree. C., and the concentration of peptide required
for red blood cell lysis of more than 80% of cells (i.e., visible lysis)
was determined at 4 and 24 hours (Table 7).
TABLE 7
Lysis of red blood cells
Lowest concentration causing lysis
.mu.g/ml
4 hours 24 hours
Indolicidin >128 32
Indolicidin-C 32 32
CP-11 >128 >128
CP-11C >128 64
CP-13 >128 128
These data show that, while indolicidin is toxic to red blood cells at 32
.mu.g/ml, but that CP-11, CP-11C, and CP-13 are significantly less toxic
to red blood cells. Additional experiments have shown that CP-11 is also
less toxic than indolicidin to T-lymphocytes (data not shown).
Example XII
Production of Additional Antimicrobial Peptides
The protein structure determinations made above have enabled the rational
design of antimicrobial peptides, many of which exhibit increased
antimicrobial activity and decreased toxicity. Expression and purification
of various cationic peptides has previously been described (Hancock et
al., U.S. Ser. No. 08/575,052, incorporated herein by reference, and Gene,
134 (1993) 7-13). In order to recombinantly express various antimicrobial
peptides described below, oligonucleotides representing both strands of
DNA encoding the various peptides were synthesized on an Applied
Biosystems 392 DNA/RNA synthesizer using conventional methods. After
annealing of the strands of DNA, the resulting DNA fragments were cloned
into the Staphylococcus aureus expression vector pRIT5. Following
transformation of the various clones into E. coli DH5.alpha., the
recombinant plasmids were purified and sequenced and then electroporated
(Bio Rad Gene Pulser Transfection Apparatus) into S. aureus K147. Nine
peptides were expressed in this system, and their relative antimicrobial
activities were determined by measuring the antimicrobial MICs of crude
preparations of the cationic peptides against Salmonella typhimurium
14028S (Piers, et al., supra) . Relative activities based on these
measurements are presented in Table 8.
TABLE 8
Relative antimicrobial activity of recombinant peptides
Gram-negative
SEQ ID activity relative
Peptide Amino acid sequence NO: to indolicidin
Indolicidin-R ILPWKWPWWPWRR 16 1X
CP-1R ILKPWKWPWWPWRR 3 0.5X
CP-3R ILPWKKWPWWRWRR 4 4X
CP-4R ILKKWPWWPWRR 5 0.5X
CP-pM ILPWKWPWRR 10 0.2X
CP-pM1b ILPWKWFFPPWPWRR 21 0.2X
CP-pM2a ILPWKWPWWPWWPWRR 11 1.5X
CP-pM2b ILPWKWPPWPPWPWRR 22 1.5X
CP-pM5 ILPWKWPWWPWWKKPWRR 12 0.5X
Methods used to express and purify cationic and microbial peptides as
protein A fusion proteins have been described (Piers et al., Gene, 134
(1993) 7-13). Here, culture supernatants of cells containing pRIT5
expressing various antimicrobial peptides were passed over an IgG
Sepharose column, according to conventional protocols. The expressed
protein then was eluted in 0.5 M acetic acid to give a relatively pure
sample. Following lyophilization, the sample was resuspended in 70% formic
acid containing 1M cyanogen bromide. Digestion was performed overnight
under nitrogen in light sealed tubes. The reaction was stopped by a ten
fold dilution in sterile distilled water, and the sample was lyophilized.
The peptide was further purified by passing the sample over a Bio-gel P100
gel sieving column, and fractions were analyzed by UV absorbance at 280 nm
and acid-urea Page. All fractions containing pure peptide were pooled and
lyophilized. The peptides were resuspended in sterile distilled water and
stored at -80.degree. C.
In a MIC assay, the peptides CP-3R, CP-pM2a and CP-pM2b had increased
activity against gram-negative bacteria when compared with indolicidin-R
(i.e., Recombinant indolicidin). The remaining peptides had the same or
decreased activity when compared with indolicidin-R. Indolicidin-R and
CP-3R subsequently were purified, and the MIC data is summarized in Table
9. CP-3R had increased activity against E. coli, P. aeruginosa, and S.
typhimurium.
TABLE 9
MIC values for recombinant peptides
Organism Indolicidin-R CP-3R
E. coli UB1005 32 8
P. aeruginosa K799 >32 8
S. typhimurium 14028S 16 8
S. aureus RN4220 16 8
S. epidermidis C621 16 4
C. albicans C627 >32 >32(8)
*Partial killing was observed in dilutions down to 8 .mu.g/ml
Example XIII
Peptide Design
Based upon the structure and function analysis of antimicrobial cationic
peptides summarized above, Applicants have devised a formula that can be
used to produce additional cationic peptides. Peptides having an amino
acid sequence encompassed by one of the formulas shown below (shown using
the one-letter amino acid code; SEQ ID NO: 23-26) are expected to have
antimicrobial activity.
X.sub.1 X.sub.1 PX.sub.2 X.sub.3 X.sub.2 P(X.sub.2 X.sub.2 P).sub.n
X.sub.2 X.sub.3 (X.sub.5).sub.o ; (SEQ ID NO: 23)
X.sub.1 X.sub.1 PX.sub.2 X.sub.3 X.sub.4 (X.sub.5).sub.r PX.sub.2
X.sub.3 X.sub.3; (SEQ ID NO: 24)
X.sub.1 X.sub.1 X.sub.3 (PW).sub.u X.sub.3 X.sub.2 X.sub.5 X.sub.2
X.sub.2 X.sub.5 X.sub.2 (X.sub.5).sub.o ; and (SEQ ID NO: 25)
X.sub.1 X.sub.1 X.sub.3 X.sub.2 P(X.sub.2 X.sub.2 P).sub.n X.sub.2
(X.sub.5).sub.m ; (SEQ ID NO: 26)
wherein:
m is 1 to 5;
n is 1 or 2;
o is 2 to 5;
r is 0 to 8;
u is 0 or 1;
X.sub.1 is Isoleucine, Leucine, Valine, Phenylalanine, Tyrosine, Tryptophan
or Methionine;
X.sub.2 represents Tryptophan or Phenylalanine
X.sub.3 represents Arginine or Lysine;
X.sub.4 represents Tryptophan or Lysine; and
X.sub.5 represents Phenylalanine, Tryptophan, Arginine, Lysine, or Proline.
In accordance with the formula provided above, Applicants have designed a
series of cationic peptides expected to have antimicrobial activity. These
peptides are listed in Table 10. This list is not exhaustive of the
antimicrobial peptides of the invention. Those skilled in the art will
recognize that, following the above formula, additional antimicrobial
cationic peptides can readily be synthesized. If desired, the
antimicrobial activity of such peptides can be measured in conventional
assays (e.g., MIC assays). Because Applicants have identified the
structural components necessary for antimicrobial activity, it is expected
that essentially all of the peptides encompassed by the formula will have
antimicrobial activity. It is not necessary that these peptides have
activity equivalent to, or better than, the peptides exemplified herein,
provided that the peptide has a detectable level of activity (e.g., in a
MIC assay).
TABLE 10
CP-14 ILKKWPWWPWKRR (SEQ ID NO: 17)
CP-15 ILKKWPWWRWRR (SEQ ID NO: 27)
CP-17 ILKKFPFFPFKKK (SEQ ID NO: 29)
CP-18 ILKKWAWWPWRRK (SEQ ID NO: 30)
CP-19 ILKKWPWWAWRRK (SEQ ID NO: 31)
CP-20 ILKKWPWWPWKKK (SEQ ID NO: 32)
CP-21 ILRRWPWWPWRRR (SEQ ID NO: 33)
CP-22 WWKKWPWWPWRRK (SEQ ID NO: 34)
CP-23 FFKKWPWWPWRRK (SEQ ID NO: 35)
CP-25 FFKKFPFFPFKKK (SEQ ID NO: 37)
CP-26 ILKKWPWWPWWPWRRK (SEQ ID NO: 38)
CP-27 ILKKWPWWPWRWWRR (SEQ ID NO: 39)
CP-28 ILKKWPWWPWRRWWK (SEQ ID NO: 40)
CP-29 ILKKWPWWPWPPRRK (SEQ ID NO: 41)
CP-30 ILKKWPWWPWPPFFRRK (SEQ ID NO: 42)
Example XIV
Synthesis and Expression of a Library Encoding Peptide Variants
A library of peptide variants was created in S. aureus using the expression
vector pRIT5. DNA strands of a combinatorial library of DNAs encoding
indolicidin variants were synthesized on the Applied Biosystems Canada
Incorporated (Ontario, Canada) 392 DNA/RNA synthesizer. These
oligonucleotides were constructed in 3 sections. The first section, the 3'
end containing restriction endonuclease sites and stop sequences, was
synthesized using standard protocols (Applied Biosystems Canada Inc.). The
second section, encoding the indolicidin peptide sequence, was synthesized
using nucleotide stocks that were doped with each of the other three
nucleotides. The relationship between the doping concentration and the
mutation rate is defined by an equation described by McNeil and Smith
(Mol. Cell. Biol., 5 (1985) 3545-3551). The third section also was
synthesized according to standard protocols (Applied Biosystems Canada
Inc.). The resulting oligonucleotides contained a pool of randomly mutated
indolicidin sequences flanked by conserved 3' and 5' sequences. The
oligonucleotide pool was then made double stranded by PCR using primers
designed to hybridize to the 3' and 5' sequences. The PCR reaction was
performed in 100 .mu.l using the conditions and parameters shown below.
PCR Reaction Parameters
1 minute 94.degree. C., 1 minute 50.degree. C., 1 minute 72.degree. C.
Cycle repeated 30 times
PCR Reaction Conditions
Final concentration
10X PCR reaction buffer 1X
10X dNTPs 1X
Taq polymerase 1 unit
Primer 1 (GCATATCGAATTCCATG) 40 ng (SEQ ID NO: 43)
Primer 2 (CTGCAGGTCGACGAGC) 40 ng (SEQ ID NO: 44)
MgCl.sub.2 10 mM
DNA Template 100 ng
The amplified product was digested at restriction endonuclease sites
located in the conserved flanking regions of the DNA fragment, and the
product was agarose gel purified using the Mermaid Glassfog (BIO 101, La
Jolla, Calif. US) DNA purification kit. After cloning the DNA fragments
into pRIT5, the randomly mutated pool of recombinant plasmids was
electroporated into S. aureus K147, and the clones were stored as a
library at -80.degree. C. Screening of a number of recombinant S. aureus
strains showed that 75% of the recombinant strains expressed the protein
A/peptide fusion product. Sequencing of a random selection of clones
revealed 4 to 8 base pair changes that resulted in 4 to 6 amino acid
substitutions (see below).
Indolicidin ILPWKWPWWPWRR (SEQ ID NO: 16)
Variant 1 ILPWICPWRPSKAN (SEQ ID NO: 13)
Variant 2 IVPWKWTLWPWRR (SEQ ID NO: 14)
Variant 3 TLPCLWPWWPWSI (SEQ ID NO: 15)
This method can be used to produce a recombinant peptide library expressing
peptides with a predetermined mutation rate.
Example XV
Screening of the Recombinant Peptide Library
A screening protocol was developed to detect an increase in antibacterial
activity in clones expressing random variants of the peptides produced as
described above. This protocol allows one to screen large numbers of
recombinant clones.
In this method, 100 ml LB high salt broth (containing 10 .mu.g/ml
chloramphenicol) was inoculated with 1 ml of an overnight culture of a
candidate clone. The culture was grown to OD.sub.600 at 37.degree. C.
while shaking at 180 rpm. Following centrifugation at 2,500 rpm for 10
minutes, the supernatant was removed and passed over a 1 ml IgG sepharose
column (Pharmacia). The fusion protein was eluted from the column with 0.5
M acetic acid (pH 3.0), and the second 1 ml aliquot contained virtually
all of the fusion protein.
The 1 ml aliquot was then lyophilized and resuspended in 75 .mu.l 70%
formic acid containing 1M cyanogen bromide. The reaction was light sealed
under nitrogen in a 1.5 ml screw top tube and rotated overnight. Following
a ten fold dilution in sterile distilled water, the reaction was again
lyophilized, washed in 200 .mu.l sterile distilled water, re-lyophilized,
and resuspended in 100 .mu.l no salt LB broth.
The peptide preparation was used in a modified broth MIC assay using a 96
well microtiter plate. A 100 .mu.l aliquot of LB no salt broth was placed
into each well. The 100 .mu.l peptide preparation was added to the first
well, and a doubling, dilution series performed through 8 wells. An
inoculum of 10.sup.2 to 10.sup.3 Salmonella typhimurium 14028S (a defensin
supersusceptible mutant) was added to each well, and the plate was
incubated overnight at 37.degree. C.
The microtiter plate was examined after 24 hours. The extract from S.
aureus strain K148 (Piers et al., supra) that contained pRIT5 and lacked
an indolicidin insert exhibited no antibacterial activity. However,
strains expressing indolicidin R and CP-R3 prevented growth of the test
organism in the first, and first and second, wells, respectively.
Therefore, peptides capable of killing at dilutions higher than 0.5 can be
identified as having an increased antimicrobial activity relative to the
reference protein. This method also can be used to identify peptides
having antimicrobial activity that is equivalent to, or less than, the
reference protein.
Example XVI
Induction of TNF in RAW 264.7 Macrophage Cells
The physiological mechanism(s) by which endotoxin exerts its effect on
humans involves the release of inflammatory cytokines, particularly tumor
necrosis factor (TNF). CP-11 was tested for its ability to block the
induction of TNF by binding LPS. This was tested in vitro in a murine RAW
264.7 macrophage cell line and in vivo for ability to reverse death in a
murine endotoxic shock model and in a P. aeruginosa infection model.
The effect of CP-11 and indolicidin on LPS-induced TNF in macrophages was
examined using the murine RAW 264.7 macrophage cell line, which produces
TNF in response to LPS. The cell line was grown by seeding 10.sup.6 cells
into a 162 cm.sup.2 cell culture flask, which was incubated at 37.degree.
C. in 5% CO.sub.2 for 1 week. RAW cell media [Dulbecco's Modified Eagle
Medium with Hepes buffer 450 ml (2.4 mM); L-glutamine 3 ml (400 mM);
Pen/Strep 3 ml (10.sup.4 U/ml of Pen, 1 mg/ml strep); and 10% heat
inactivated fetal bovine serum (FBS) 50 ml] was then removed from the cell
culture flasks. A 10 ml aliquot of solution was added to each flask and
incubated at 37.degree. C. for 10 minutes. The cells were removed from the
flasks, diluted in RAW cell media, and centrifuged for 6 minutes. The cell
pellet was resuspended in 5 ml of media/flask. A 100 .mu.l cell suspension
was removed and added to 400 .mu.l of trypan blue, and the cells were
counted using a hemocytometer. The cell suspension was diluted to
1.times.10.sup.6 cells/ml and 1 ml of suspension was added per well of a
24 well plate. The 24 well plates were incubated at 37.degree. C. in 5%
CO.sub.2 overnight for use in the assay.
After an overnight incubation, the media was aspirated from all the wells.
LPS was added at 100 ng/100 .mu.l. Peptide was added at the desired
concentration/100 .mu.l to specified wells. RAW cell media was added to
all the wells so they had a final volume of 1 ml. The plates were then
incubated for six hours at 37.degree. C. in 5% CO.sub.2. The supernatant
was then removed from the wells and stored overnight at 4.degree. C.
Experiments were also performed using whole bacteria in a transwell filter
system. Viable Bort E. coli and P. aeruginosa were incubated in 0.22 .mu.m
filter inserts, which prevented direct contact of the bacteria with the
macrophage cells yet allowed products released by the bacteria (e.g., LPS)
to interact with the cells. Overnight cultures were diluted in phosphate
buffered saline to an OD.sub.600 of 0.3 (approximately 10.sup.8 cells/ml).
The bacteria were further diluted 1:100 and counted.
The supernatants from the above tissue culture experiments were used in the
cell cytotoxic L929 assay. The RAW cell supernatants were diluted in a
three fold dilution series in 96 well plates. A 50 .mu.l aliquot of TNF
media was added to all of the wells in all of the plates except to those
wells in the first row. Ten .mu.l of murine TNF standard (20 ng/ml) and 90
.mu.l of TNF media were added in duplicate to the plate and diluted 1:2
down the plate to the second to last row.
TNF-sensitive L929 mouse fibroblast cells were seeded at 10.sup.6 cells/162
cm.sup.2 cell culture flask and left to grow for 1 week. L929 cells were
removed from the flask with 10 mls of trypsin EDTA/flask and incubated 3-5
minutes. The cell suspension was diluted, and then centrifuged for 6
minutes. The pellet was resuspended in 5 mls of fresh L929 media/flask and
counted (same as RAW cells). Cell suspension was diluted to 10.sup.6
cells/ml. One hundred .mu.l was used to inoculate each well of the 96 well
plates with the supernatants. (L929 Growth Media was the same as RAW cell
media except, instead of FBS, 50 mls of 10% heat inactivated horse serum
was utilized; TNF Assay Media was the same as RAW cell media except 4
.mu.g/ml Actinomycin D.) The plates were incubated for two days at
37.degree. C., 5% CO.sub.2. The plates were then read at 570 nm in a ELISA
plate reader with 690 nm reference filter. One unit of TNF activity was
defined as the amount required to kill 50% of the L929 cells. The TNF
level in Units per ml therefore was the reciprocal of the dilution that
led to a 50% killing of L929 cells. Computations were performed using the
ELISA+ program.
FIG. 9 shows levels of TNF (U/ml) produced by the macrophage cells after a
6 hour treatment with increasing amounts (0, 2, 5, 10, 20, or 50 .mu.g) of
either CP-11 or indolicidin peptide and 100 ng of E. coli 0111:B4 LPS. The
data indicates that both peptides efficiently reduced the level of
LPS-induced in macrophages by LPS by 70% and 71 % respectively. The
results of a 6 hour incubation with P. aeruginosa LPS and indolicidin or
CP-11 are shown in FIG. 10. Fifty .mu.g of CP-11 and indolicidin decreased
the TNF production by 51% and 67% respectively. Fifty .mu.g of indolicidin
and PP-11 decreased TNF production by macrophages incubated with 100 ng
Bort E. coli LPS by 68% and 74% respectively.
In the experiments using intact bacteria, the peptides were able to
effectively reduce the induction of TNF by the diffusible products of P.
aeruginosa and E. coli. CP-11 and Indolicidin reduced the TNF-induction by
LPS released by P. aeruginosa by 97% and 91% respectively (FIG. 11). Both
peptides reduced the TNF-induction by Bort E. coli by 99% (FIG. 12).
To confirm that indolicidin was acting on LPS rather than directly upon
macrophage cell lines, 20 .mu.g of indolicidin was added to RAW cells and
incubated for 60 minutes prior to aspiration of the medium and washing the
cells 3 times with HBSS (Hanks Buffered Salt Solution). Addition of 100 ng
of LPS to the washed RAW cells resulted in a high level of TNF induction
(4168 Units of TNF per ml), suggesting that the indolicidin had not
permanently depressed the ability of RAW cells to induce TNF in response
to LPS addition. In contrast, the aspirated medium containing indolicidin
could depress the ability of fresh RAW cells to induce TNF in response to
100 ng of E. coli Bort LPS by 74%. Up to 50 .mu.g of CP-11 or indolicidin
caused no apparent decrease in Raw cell viability as judged by Trypan blue
exclusion.
Example XVII
Murine Endotoxic Shock Model
The ability of indolicidin and variants to protect against LPS-induced
endotoxemia was assessed in vivo. Mice (8-10 weeks old) were injected
intraperitoneally (IP) with 20 mg D-galactosamine (Dgal) to sensitive them
to LPS according to the model of Galanos (Galanos, C., M. A., Freudenberg
and W. Reutter, 1979, Galactosamine sensitization to the lethal effects of
endotoxin. Proc. Natl. Acad. Sci. USA 76:5939), followed by 200 .mu.g
peptide in 100 .mu.l. Immediately afterwards LPS (10 .mu.g) in 100 .mu.l
was injected. The mice were observed 24 hours after injections and
survivors noted. Injection of 200 .mu.g of indolicidin-D, CP-11, or CP-11,
or CP-11D did not cause mortality. When Dgal and LPS were also injected,
that 200 .mu.g of indolicidin reduced mortality reduced at 24 hours from
100% in controls to 60%. This example thus demonstrates that the cationic
peptides of the invention have anti-endotoxin activity in vivo.
Although the invention has been described with reference to the presently
preferred embodiment, it should be understood that various modifications
can be made without departing from the spirit of the invention.
Accordingly, the invention is limited only by the following claims.
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